title: Cloning, expression, and characterization of serine protease from thermophilic fungus Thermoascus aurantiacus var. levisporus.
abstract: The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.
authors.affiliation: Department of Environmental Biology, Shandong Agricultural University, Tai'an, Shandong, 271018, PR China.
authors.forename: An-Na
authors.initials: AN
authors.lastname: Li
authors.forename: Chen
authors.initials: C
authors.lastname: Xie
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Jia
authors.initials: J
authors.lastname: Zhang
authors.forename: Duo-Chuan
authors.initials: DC
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D055327:Thermoascus / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D057057:Serine Proteases